Biological Applications
8.2 Pharmaceutical Applications
Infrared spectroscopy has been extensively used in both qualitative and quantita-tive pharmaceutical analysis [1–3]. This technique is important for the evaluation of the raw materials used in production, the active ingredients and the excipients (the inert ingredients in a drug formulation, e.g. lactose powder, hydroxypropyl cellulose capsules, etc.). Although nuclear magnetic resonance spectroscopy and mass spectrometry are widely used in the pharmaceutical industry for the identi-fication of drug substances, infrared spectroscopy can provide valuable additional structural information, such as the presence of certain functional groups.
Figure 8.1 illustrates the diffuse reflectance infrared spectrum of acetylsalicylic acid (aspirin). Such a spectrum may be used to identify the functional groups present in this molecule. The presence of O–H groups in acetylsalicylic acid is indicated by a broad band in the 3400–3300 cm−1 region and C–H stretching bands overlap with this band in the 3000–2800 cm−1range. The spectrum shows two strong C=O stretching bands at 1780 and 1750 cm−1, so indicating that the molecule contains carbonyl groups in different environments. The spectrum also shows strong bands at 1150 and 1100 cm−1 due to C–O stretching: the 1150 cm−1band is due to the presence of a C–O–H group, while the 1100 cm−1 band is due to a C–O–C group in the structure. There is also evidence of a benzene ring, including a series of characteristic bands in the 800–500 cm−1 range due to C–H stretching in the aromatic ring. In summary, acetylsalicylic acid contains O–H, C=O, C–O–C, C–O–H and aliphatic and aromatic C–H groups.
The acetylsalicylic acid structure is illustrated in Figure 8.2, hence confirming the presence of these functional groups.
4000 3000 2000
Wavenumber (cm−1)
1000
Intensity
Figure 8.1 Diffuse reflectance infrared spectrum of acetyl salicylic acid. From Stuart, B., Biological Applications of Infrared Spectroscopy, ACOL Series, Wiley, Chichester, UK, 1997. University of Greenwich, and reproduced by permission of the University of Greenwich.
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COOH
OCCH3 O
Figure 8.2 Structure of acetylsalicylic acid.
COOH
OH Figure 8.3 Structure of salicylic acid (cf. DQ 8.1).
DQ 8.1
Salicylic acid is a compound used to synthesize aspirin. The structure of this compound is shown below in Figure 8.3. What differences would be expected in the infrared spectra of salicylic acid and acetylsalicylic acid?
Answer
A comparison of the molecular structures of salicylic acid and acetylsal-icylic acid informs us that the difference between these two molecules is the presence of an O–H group in the salicylic acid and an acetyl O–COCH3 group in the acetylsalicylic acid. The crucial differences in the spectra will manifest themselves in the O–H stretching and C=O stretching regions of each spectrum.
SAQ 8.1
The structure of the steroid hormone testosterone is illustrated below in Figure 8.4, with the infrared spectrum of a Nujol mull of testosterone being shown below in Figure 8.5. Assign the major infrared bands of testosterone.
O
CH3
CH3 OH
Figure 8.4 Structure of testosterone (cf. SAQ 8.1).
Another important aspect of pharmaceutical analysis is the characterization of the different crystalline forms (polymorphs) of pharmaceutical solids. It is well established that the different forms of a drug can exhibit significantly different physical and chemical properties, and often one form is preferred due to its superior drug activity. An example of how the differences between
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20 40 60 80 100
Transmittance (%)
Wavenumber (cm−1)
Figure 8.5 Infrared spectrum of testosterone (cf. SAQ 8.1). Reprinted from Spectrochim.
Acta, 43A, Fletton, R. A., Harris, R. K., Kenwright, A. M., Lancaster, R. W., Pacher, K. J. and Sheppard, N., ‘A comparative Spectroscopic investigation of three pseudopoly-morphs of testosterone using solid-state IR and high resolution solid-state NMR’, 1111 – 1120, Copyright (1987), with permission from Elsevier.
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Wavenumber (cm−1)
Intensity
(a)
(b)
Figure 8.6 Diffuse reflectance infrared spectra of (a) form I and (b) form II of cortisone acetate [4]. Reprinted from Spectrochim. Acta, 47A, Deeley, C. M., Spragg, R. A. and Threlfall, T. L., ‘A comparison of Fourier transform infrared and near-infrared Fourier transform Raman spectroscopy for quantitative measurements: an application in polymor-phism’, 1217 – 1223, Copyright (1991), with permission from Elsevier.
polymorphs are observed in their infrared spectra is illustrated in Figure 8.6, in which the diffuse reflectance spectra of two of the polymorphic forms (I and II) of the anti-inflammatory agent, cortisone acetate, are shown [4]. The structure of cortisone acetate is shown in Figure 8.7. Diffuse reflectance is a suitable
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O
H3C
CH3 CH3 O
O
O OH
Figure 8.7 Structure of cortisone acetate.
sampling technique because it avoids the structural changes that may occur under high pressures during the formation of the alkali halide discs. Although the spectra shown in Figure 8.6 are complex below 1500 cm−1, in the region above 1500 cm−1 it is easier to identify differences in the spectra of the two polymorphs. In the 1800–1500 cm−1 region, there are significant differences in band intensities. There is also a notable difference in the appearance of the O–H stretching band in the 3600–3100 cm−1 region.
SAQ 8.2
An anti-inflammatory agent, the structure of which is shown below in Figure 8.8, forms two crystalline polymorphs (I and II) that have been differentiated by microscopic techniques and show different properties [5]. The carbonyl regions of the diffuse reflectance spectra of the two polymorphs are shown below in Figure 8.9. Comment on the differences in the carbonyl regions in the spectra of these polymorphs. Suggest a simple method for quantitatively determining the amount of polymorph II in a mixture of the two polymorphs of this drug.
O O O
CH3O
CH3
O
CH3 CH3
COOH (±)
Figure 8.8 Structure of an anti-inflammatory agent (cf. SAQ 8.2) [5]. Reprinted from J.
Pharmaceut. Biomed. Anal., 11, Roston, D. A., Walters, M. C., Rhinebarger, R. R. and Ferro, L. J., ‘Characterization of polymorphs of a new anti-inflammatory drug’, 293 – 300, Copyright (1993), with permission from Elsevier.
1800 1750 1700 1650 1600 1550 1500 Wavenumber (cm−1)
0 0.2 0.4 0.6 0.8 1.0
1760 1645
(a)
1800 1750 1700 1650 1600 1550 1500 Wavenumber (cm−1)
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
AbsorbanceAbsorbance 1740 1710 1670
(b)
Figure 8.9 Carbonyl regions of the diffuse reflectance infrared spectra of two poly-morphs of an anti-inflammatory drug: (a) polymorph I; (b) polymorph II (cf. SAQ 8.2) [5]. Reprinted from J. Pharmaceut. Biomed. Anal., 11, Roston, D. A., Walters, M. C., Rhinebarger, R. R. and Ferro, L. J., ‘Characterization of polymorphs of a new anti-inflammatory drug’, 293 – 300, Copyright (1993), with permission from Elsevier.
Near-infrared (NIR) spectroscopy lends itself to the pharmaceutical quality control laboratory [6, 7]. The development of fibre optic probes for remote anal-ysis has lead to the expansion of its use in the pharmaceutical industry. Libraries of the NIR spectra of compounds commonly used in the pharmaceutical industry are available. Quantitative analysis can also be effectively carried out by using multivariate techniques such as principal component analysis (PCA).
Gas chromatography–infrared (GC–IR) spectroscopy is an appropriate tech-nique for drug analysis as it can be used for isomer separation or contaminant detection. Amphetamines are one class of drug that have been successfully differ-entiated by using GC–IR spectroscopy. Amphetamines are structurally similar molecules that can be easily mis-identified. Although such similar compounds cannot be differentiated by their mass spectra, there are prominent differences
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Wavenumber (cm−1)
Absorbance
(a)
(b)
(c)
(d)
Figure 8.10 Infrared spectra of the components of a clandestine laboratory drug mixture separated by GC– IR spectroscopy: (a) ephedrine; (b) pseudoephedrine; (c) amphetamine;
(d) methamphetamine [8]. From Bartick, E. G., ‘Applications of Vibrational Spectroscopy in Criminal Forensic Analysis’, in Handbook of Vibrational Spectroscopy, Vol. 4, Chal-mers, J. M. and Griffiths, P. R. (Eds), pp. 2993 – 3004. Copyright 2002. John Wiley &
Sons Limited. Reproduced with permission.
NH2
CH3
(a) H
N
CH3
(b)
CH3
Figure 8.11 Structures of (a) amphetamine and (b) methamphetamine.
in their infrared spectra. Figure 8.10 illustrates the gas-phase FTIR spectra of a clandestine laboratory mixture separated by GC–IR spectroscopy. The spectra of amphetamine and methamphetamine are illustrated and the structures of these two compounds are shown in Figure 8.11. The spectra importantly differ in the region below 1700 cm−1[8]. The N–H bending band near 1600 cm−1 is more intense for amphetamine, which is a primary amine.
SAQ 8.3
The stereoisomers, ephedrine and pseudoephedrine, are precursors for metham-phetamine. The gas-phase infrared spectra of these compounds, separated by using GC–IR spectroscopy, are shown in Figure 8.10. From inspection of these spectra, is it possible to differentiate these compounds in a forensic sample obtained from a clandestine laboratory?